LABORATORY STANDARD OPERATING PROCEDURE ......GenomeLab Separation Buffer, 4x30 ml (Beckman Coulter,...

LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET

MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC

O157) – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

CODE: PNL19

Effective Date:

09 14 10

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 20

1. PURPOSE: to describe the standardized laboratory protocol for molecular subtyping of Shiga

toxin-producing Escherichia coli O157 (STEC O157).

2. SCOPE: to provide the PulseNet participants with a single protocol for performing MLVA of

STEC O157, thus ensuring inter-laboratory comparability of the generated results using the

Beckman Coulter CEQ 8000/8800/GeXP platform.

3. DEFINITIONS: 3.1. MLVA: Multiple-locus variable-number tandem repeat analysis

3.2. VNTR: Variable-number tandem repeat

3.3. DNA: Deoxyribonucleic acid

3.4. DNase: Deoxyribonuclenase

3.5. PCR: Polymerase chain reaction

3.6. HPLC: High purity liquid chromatography

3.7. dNTP: Deoxyribonucleotide triphosphate

3.8. CDC: Centers for Disease Control and Prevention

4. RESPONSIBILITIES/PROCEDURE 4.1. Biosafety warning: Shiga toxin-producing Escherichia coli O157 (STEC O157) is a

human pathogen causing serious disease. It has been reported that less than 100 cells of

STEC O157 may cause infection. Always use a minimum of Biosafety level 2 practices

and extreme caution when transferring and handling strains of this serotype. Work in a

biological safety cabinet when handling large amounts of cells. Disinfect or dispose of all

plastic ware and glassware that come in contact with the cultures in a safe manner.

4.2. Reagents, supplies and equipment needed for DNA template preparation

4.2.1. Trypticase soy agar with 5 % sheep blood (TSA-SB) or comparable media

4.2.2. 1 µl and 10 µl inoculation loops

4.2.3. 0.5 ml microcentrifuge tubes

4.2.4. DNase-free, molecular biology -grade water

4.2.5. Vortex

4.2.6. Boiling water bath or thermal block / thermocycler accommodating 0.5 ml tubes

4.2.7. Tabletop centrifuge for high rpm (up to 13,000-14,000 rpm) spinning

4.3. Reagents, supplies and equipment needed for PCR

4.3.1. DNA lysates from isolates (store at -20oC or -80ºC freezer for long term)

4.3.2. PCR primers (see appendix PNL19-1)

4.3.2.1. Fluorescent-labeled forward primers

4.3.2.1.1. HPLC-purified

4.3.2.2. Unlabeled reverse primers

4.3.2.2.1. Regular gel filtration purification

4.3.2.3. Integrated Technologies (Skokie, IL; www.idtdna.com, 1-800-

328-2661) and Proligo (Boulder, CO; www.proligo.com, 1-800-234-

5362) are currently the suppliers for the Wellred dye-labeled (D2)

primers recommended by Beckman Coulter. Biosearch Technologies

(Novato, CA; www.biosearchtech.com; 1-800-436-6631) is the

http://www.idtdna.com/

http://www.proligo.com/




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recommended supplier for the Quasar dye –labeled (Quas670 and

705) primers.

4.3.2.4. Divide the concentrated stocks (100 µM) in portions and store at

-80ºC freezer

4.3.2.4.1. One vial should contain enough to prepare 25-50 µl of

working solution. Avoid repetitive freeze-thaw cycles of

concentrated primer stocks.

4.3.2.5. The 1 µM, 2.5 µM, 5 µM and 25 µM working solutions can be

stored at either -20oC or -80ºC freezer

4.3.2.6. Prepare new working solutions every month or if a significant

drop in the fluorescence level is observed (for instructions refer to

PNQ05_MLVA CEQ certification, appendix PNQ05-5)

4.3.3. 96-well polypropylene PCR plates (Fisher, Cat. No. 07-200-613) or Microamp

PCR tubes without caps (Life Technologies, Cat. No. N8010533)

4.3.4. 8-well strip caps for polypropylene plate (Fisher, Cat. No. 07-200-639) or

MicroAmp strip caps for individual tubes (Life Technologies, Cat. No.

N8010535)


4.3.6. 1.5 ml Eppendorf microcentrifuge tubes

4.3.7. PCR Nucleotide mix (ready-to-use dNTP mix containing all four nucleotides;

Roche, Cat. No. 11 814 362 001)

4.3.8. Platinum Taq Polymerase with 50 mM MgCl2 and 10X buffer (Life

Technologies Cat. No. 10966-034)

4.3.9. PCR cooling block (VWR International, Cat. No. 62111-762)

4.3.10. DNA Engine (Biorad), GeneAmp (Life Techologies) or similar thermocycler

with a heated lid option and 96-well block format

4.3.11. Parafilm M, 4” width (VWR, Cat. No. 52858-032)

4.3.12. Complete set (1000 µl, 200 µl, 100 µl, 20 µ, 10 µl, and 2 µl) of single channel

pipettors for mastermix set-up (“clean set”)

4.3.13. A 1-10 µl single channel pipettor for adding DNA templates

4.3.14. Filtered tips for pipettors

4.3.15. Microfuge for low (up to 6,000 rpm) rpm spinning

4.4. Reagents, supplies and equipment needed for Beckman CEQ 8000/ 8800/GeXP)


4.4.2. PCR cooling block (VWR International, Cat. No. 62111-762)

4.4.3. 10 l, 100 l, and 1000 l single channel pipettors

4.4.4. 1-10 l and 20-200 µl multichannel pipettors

4.4.5. Filtered pipette tips

4.4.6. Sterile solution basins

4.4.7. 1.5 ml Eppendorf microcentrifuge tubes

4.4.8. 5 ml polystyrene round bottom tubes (for example Falcon tube 352058)

4.4.9. 96-well polypropylene (non-PCR) V-bottom plate (for dilutions; Fisher

Scientific, Cat. No. 07-200-698)




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4.4.10. 96-well Beckman sample plates (Beckman Coulter, Cat. No. 609801;

alternative: Fisher Scientific, Cat. No. 07-200-613)

4.4.11. 96-well Beckman CEQ buffer plate (Beckman Coulter, Cat. No. 609844;

alternative: Fisher Scientific, Cat. No. 07-200-98)

4.4.12. Mineral oil (molecular grade) (Sigma, Cat. No. M5904)

4.4.13. GenomeLab Sample Loading Solution (Beckman Coulter, Cat. No. 608082)

4.4.13.1. After first thawing, divide in portions of 1.0 ml / vial in order to

avoid repeated freeze-thaw cycles

4.4.14. GenomeLab DNA size standard kit 600 bp (Beckman Coulter, Cat. No.

608095)

4.4.14.1. After first thawing, divide in portions of 8 µl / vial in order to

avoid repeated freeze-thaw cycles

4.4.15. GenomeLab Fragment Analysis Test Sample (Beckman Coulter, Cat. No.

608105)

4.4.15.1. Needed only once to establish the system dye color spectra for

the instrument

4.4.15.2. Useful also for troubleshooting

4.4.16. GenomeLab Separation Capillary Array (Beckman Coulter, Cat. No. 608087)

4.4.17. GenomeLab Separation Buffer, 4x30 ml (Beckman Coulter, Cat. No. 608012)

4.4.18. GenomeLab Separation Gel LPAI (Beckman Coulter, Cat. No. 608010 for

CEQ 8000/single rail GeXP; Cat. No. 391438 for CEQ 8800/double rail GeXP)

4.4.19. Parafilm M, 4” width (VWR, Cat. No. 52858-032)

4.4.20. A rinse bottle containing distilled water

4.4.21. Centrifuge with a microtiter plate rotor

4.4.22. Microfuge for low (up to 6,000 rpm) rpm spinning

4.5. Isolate preparation

4.5.1. Day 0

4.5.1.1. Streak an isolated colony from pure test cultures to TSA-SB

plate (or comparable media). Incubate cultures at 37ºC for 14-18 hrs.

4.5.2. Day 1

4.5.2.1. For each isolate to be typed, aliquot 100 µl of sterile, molecular

biology-grade water into 0.5 ml microfuge tubes. Use a sterile,

disposable 1 µl loop to pick 2-3 colonies (about half of a loopfull);

rotate the loop in the microfuge tube to release the bacteria into the

water. Cap and vortex for 10-15 seconds to disperse any clumps.

4.5.2.2. Place the tubes in a 100ºC water bath or heat block for 10

minutes. Cool briefly on ice or in fridge and centrifuge for 10

minutes at 10,000 rpm. Place on ice or in fridge while preparing

PCR reactions. These DNA templates can be stored at -20oC or -80ºC

for several years.

4.6. PCR procedure

4.6.1. Day 1

4.6.1.1. Fill out, save with the run name, and print a PCR template/CEQ

worksheet (see appendix PNL19-2) with appropriately labeled




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samples (a maximum of 43 isolates/plate; 40 unknowns + two

positive controls and a negative control; two wells are reserved for

the internal ladder; the first column of the plate is reserved for

conditioning).

4.6.1.1.1. For each isolate, two wells must be labeled as follows:

BNkeyR1 where “BNkey” represents the isolate-specific state

laboratory identification number (be sure to use the exactly same

isolate ID that is used in the PFGE gels uploaded to the national

database) and “R1” represents one of the two specific multiplex

PCR reactions (R1, R2).

4.6.1.1.2. The CEQ8000/ 8800/GeXP machine has an 8-capillary

array and must always be run with a complete column (n=8

wells). For example, 54 wells (6.75 8-well columns) are required

to test 26 samples (including controls). The remaining two wells

for the seventh column must be filled and run with 20 µl of water

or left-over sample loading solution (blanks).

4.6.1.1.3. Also save the CEQ worksheet for the run on a flash drive

(memory stick). That way you can easily import the sample plate

set-up in the CEQ without having to re-type the sample keys.

4.6.1.2. Fill out, and print a PCR mastermix calculation worksheet (see

appendix PNL19-3) by typing the number of isolates to be tested

(plus 2-3 extra) in the PCR mastermix calculators labeled R1 and R2.

This number is highlighted in red and is next to “number of samples

to be analyzed”. The mastermixes for reactions 1 and 2 (R1, R2) for

one sample are as follows:

R1 Volume ( µl) Final conc.

PCR water 5.16

PCR buffer (10x) 1.00 1x

MgCl2 (50 mM) 0.40 2.00 mM

dNTPs (10 mM) 0.20 0.20 mM

VNTR-3F (25 µM) 0.18 0.45 µM

VNTR-3R (25 µM) 0.18 0.45 µM

VNTR-34F (5 µM) 0.28 0.14 µM

VNTR-34R (5 µM) 0.28 0.14 µM

VNTR-9F (5 µM) 0.28 0.14 µM

VNTR-9R (5 µM) 0.28 0.14 µM

VNTR-25F (2.5 µM) 0.28 0.07µM

VNTR-25R (2.5 µM) 0.28 0.07 µM

Taq (5 U/µl) 0.20 1 U

= 9.00




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R2 Volume (µl) Final conc.

PCR water 5.96

PCR buffer (10x) 1.00 1x

MgCl2 (50 mM) 0.40 2.00 mM

dNTPs (10 mM) 0.20 0.20 mM

VNTR-17F (5 µM) 0.18 0.09 µM

VNTR-17R (5 µM) 0.18 0.09 µM

VNTR-19F (1 µM) 0.20 0.02µM

VNTR-19R (1 µM) 0.20 0.02 µM

VNTR-36F (1 µM) 0.12 0.012 µM

VNTR-36R (1 µM) 0.12 0.012 µM

VNTR-37F (2.5 µM) 0.12 0.03µM

VNTR-37R (2.5 µM) 0.12 0.03µM

Taq (5 U/µl) 0.20 1 U

= 9.00

4.6.1.2.1. NOTE: NOTE: these primer concentrations serve as a starting

point. Since laboratory-specific factors, such as the age of the

primer stocks, calibration status of the thermocyclers and

pipettes, etc. affect amplification efficiency, each laboratory will

have to re-optimize the primer concentrations for optimal

detection of all targets. However, any other parameters stated in

the SOP should not be changed.

4.6.1.3. Thaw all reagents and supplies needed for PCR reactions and

place on ice; keep primers light protected as much as possible.

4.6.1.3.1. PCR mastermixes should be set up in a clean hood that is

dedicated just for this purpose and where no cultures or DNA are

handled.

4.6.1.4. Prepare the two separate PCR mastermixes in 1.5 ml Eppendorf

tubes following the instructions in the PCR mastermix calculation

worksheet (see appendix PNL19-3) and place on ice. Add the

mastermix components in the following order: water, 10x PCR

buffer, Mg2Cl, dNTPs, primers, and then finally Taq polymerase.

Mix the reaction mixture by vortexing briefly.

4.6.1.4.1. Vortex all reagents except Taq polymerase before adding to

the mastermix. Taq may be briefly centrifuged with low rpm, if

necessary, to pull the enzyme down to the bottom of the tube.

4.6.1.5. Place a 96-well PCR plate or required number of PCR tubes in a

PCR cooling block.

4.6.1.6. Dispense 9.0 µl of each mastermix into the appropriate columns

of the 96-well polypropylene plate / PCR tubes as noted in the PCR

template/CEQ worksheet (see appendix PNL19-2)

4.6.1.7. Add 1 l of PCR water to each of the two different wells

representing the negative controls of the two reactions.




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4.6.1.8. Add 1.0 l of DNA lysate to each of the two different wells

representing the two PCR reactions. Use STEC O157 strain EDL933

(ATCC 43895) as a positive control (it is recommended to run the

positive control in duplicate). The internal ladder to be used will be

comprised of pooled PCR products of the isolates EC04PN0139 and

EC04PN0570 (see appendix PNL19-5 for instructions for ladder

preparation)

4.6.1.9. Cover all wells / tubes with 8-well strip caps and firmly clamp

down to avoid any evaporation during PCR amplification.

4.6.1.10. Recommendation: briefly spin down the plate / tubes to remove

any air bubbles.

4.6.1.11. Setup the following PCR parameters and save the program as

“O157MLVA”:

* 95ºC for 5 min Step 1

* 94ºC for 20 sec Step 2



* Go to step 2, 34x Step 5

* 72ºC for 5 min Step 6

* Indefinite hold at 4ºC

4.6.1.11.1. Make sure to use the heated lid option on the PCR block

and “calculated” or tube control (instead of block or probe control)

as a temperature control method.

4.6.1.12. When the PCR is complete, store the amplification products

light-protected at 4ºC until ready to run on CEQ/GeXP. If the

fragment analysis is not performed the same day, the plate should be

stored at -20oC or -80ºC. The PCR products are stable for

approximately one month, when stored frozen.

4.7. Initial setup of CEQ8000/ 8800/GeXP instrument 4.7.1. NOTE: Steps 4.7.2.-4.7.8. only need to be performed before the very first run

and every time a new working database is created.

4.7.2. In the main page of the CEQ software version 8.0 or 9.0 or the GeXP software

version 10.2, click on the “Fragments” icon. Select “Analysis” in the bar at the

top of the window to open a window called “Analysis Parameters”.

4.7.3. Select “DefaultFragmentAnalysisParameters” in the top drop-down menu and

click “Edit”. In the appearing “Edit Fragment Analysis Parameters” window,

select the tab “Analysis Method” and select “Size Standard 600” for size standard

and “Quartic” for model. Do not make any other changes.

4.7.4. Click on “Save As” and name the analysis parameter as “MLVA600”.

4.7.5. Go back to the main page of the CEQ program and click on the “Set Up” icon.

Select “Create a New Sample Plate” and click “OK”.




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4.7.6. In the “Sample Setup” window, check to be sure that the methods “Condition”

and “Frag-test” are present in the drop-down menu below each column. These

should be the instrument default methods.

4.7.7. If they are not present or to create the frag-test_pause method, highlight any of

the default methods and select “Method” tab from below. This will open a

“Method” window. Click on the “Edit” button in this open window and put in the

following parameters in the “CEQ Methods” table below.

CEQ Methods:

Condition Frag-test_pause

Capillary Temperature 35 35

Wait 0 Yes

Denature Temperature 90 90

Time 0 120 seconds

Pause Time NA 10 min

Inject Voltage 2.0 kV 2.0 kV

Time 30 seconds 15 seconds

Separation Voltage 7.5 kV 6.0 kV

Time 35 minutes 60 minutes

Pause 0 minutes 0 minutes

4.7.8. Be sure to save the new method with new file names. Each time a new CEQ

database is generated, these methods are not copied. However, if these methods

were in the machine as a default (Frag-test_pause is not), they are copied when a

new database is created.

4.8. CEQ8000/ 8800/GeXP instrument preparation before each run

4.8.1. Day 1 4.8.1.1. Make sure a capillary array is installed in the instrument. For

installation, follow the instructions in the appendix PNL19-4.

4.8.1.1.1. If the instrument is used frequently, there is no need to remove

the array after every run. Beckman Coulter guarantees the shelf

life of the array for up to 30 days (or up to 100 runs) at room

temperature. However, as long as the positive control and the

internal ladder fall within the sizing range indicated in the

appendices PNL19-2 and PNL19-5 and the fragment analysis

failure rate is less than 5 %, the same capillary array can be used

up to 45 to 50 days.

4.8.1.2. To setup the plate run on the CEQ\GeXP, click on the “Set Up”

icon on the CEQ\GeXP main page and select “Create a New Sample

Plate” and click “OK”.

4.8.1.3. Using a USB flash drive, open the PCR/CEQ Excel worksheet

(PNL19-2) saved for this plate run and copy only the 96-well format

and paste it directly into the blank sample setup window.




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4.8.1.4. Under the first column of the plate format, select the method

“Condition” from the drop-down menu. Select the method “Frag-

test_pause” for the columns containing the samples.

4.8.1.5. Highlight all columns containing samples. At the bottom of the

screen, select the “Analysis” tab, check the box for “Perform

analysis”, and select the “MLVA600” parameter set from the drop-

down menu.

4.8.1.6. NOTE: The export tab does not contain the correct format

(CSV) and should be left unchecked.

4.8.1.7. Save the plate with a run name following the standardized

PulseNet naming system: Use the unique identifier code that was

assigned to your laboratory by PulseNet for the first two to four

letters of the file name. The next two spaces indicate the year and the

next four spaces indicate the month and the date the run was

performed. For example GA070426 is a run made at the GA Public

Health Laboratory on April 26th

2007. If several runs are performed

the same day, separate the file names by using sequential numbers,

for example GA070426-1, GA070426-2.

4.8.1.8. Close “Set Up” module.

4.8.1.9. Click on the “Run” icon in the main CEQ\GeXP window. The

“Run Control – CEQ System” window will appear.

4.8.1.10. If the instrument does not have a gel cartridge installed, the

“Instrument Warning Message” window will appear stating

“Instrument Status: Loading Gel Cartridge”. Click “OK”.

4.8.1.11. If the instrument already has a gel cartridge installed, the above

message does not appear. In this case, you can check the amount of

gel available by clicking on the “Life” tab on the left side of the “Run

Control – CEQ System” window. If a new gel cartridge needs to be

installed, select “Release Gel Cartridge” from the “Replenish” drop-

down menu or from the toolbar and wait for the CEQ\GeXP to

display a green “Go” signal box.

4.8.1.11.1. NOTE: A full plate uses approximately 9.0 ml of gel.

4.8.1.12. Open the gel access door, pull out the yellow plug or the old gel

cartridge and install the new gel cartridge. Select “Install Gel

Cartridge” from the “Replenish” drop-down menu or from the tool

bar. The “Install Gel Cartridge” dialog box will open. Type in the lot

number for the gel, click on “Set To New” to zero out the time on the

instrument (if the cartridge is new) or type in the number of hours the

gel cartridge (previously used) has spent in the instrument. For the

GeXP, designate whether the gel cartridge is ‘New’ or ‘Used’ by

selecting the corresponding radial button. If installing a previously

used cartridge, enter the number of hours on instrument. When

finished click on “Done”.

4.8.1.13. Perform a manual purge by selecting “Manifold Purge” from the

“Direct Control” drop-down menu or by clicking on the funnel on the




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direct control screen. A “Manifold Purge” dialog box will open. Set

0.1 ml as the purge volume and 2 as the number of purge cycles and

click on “Purge”.

4.8.1.13.1. NOTE: a manifold purge is only necessary when a new/used

gel cartridge is inserted. If using a cartridge already installed in

the instrument, a manifold purge is not necessary.

4.9. Fragment analysis sample preparation

4.9.1. Day 1

4.9.1.1. Thaw the CEQ sample loading solution (SLS), 600 bp DNA size

standard and internal ladder (see appendix PNL19-5 for preparation

instructions) and place on ice.

4.9.1.2. While the reagents are thawing, prepare a Beckman buffer plate

by filling a required amount of wells about ¾ full (~250 l) with

CEQ Separation Buffer.

4.9.1.3. Prepare a 96-well V-bottom plate for diluting the PCR reactions.

Using a 200 l multichannel pipettor and a solution basin, dispense

59 µl of molecular-grade water in the required number of wells.

4.9.1.4. Remove the plate / tubes with the PCR reactions from the

thermocycler. Briefly spin down the plate / tubes, if necessary. Use a

10 l multichannel pipettor to transfer 1 l of each PCR reaction

directly across to the corresponding set of wells in the dilution plate.

In order to avoid cross-contamination, remove the strip cap from just

one column at a time and recap the column before opening the next

one.

4.9.1.5. For the internal ladder, combine R1 and R2 PCR products from

the four PCR reactions of both internal ladder isolates into one tube

to end with a total of 40 µl. Mix well by pipetting up and down a few

times and add 3 l of internal ladder in two wells.

4.9.1.6. Using a 200 µl multichannel pipettor, mix the dilutions by

pipetting up and down a few times. Cover the plate with Parafilm and

put in the fridge or on ice.

4.9.1.7. Prepare a fragment analysis master mix containing DNA size

standard and SLS buffer for the samples following the calculations

indicated in the table below. The fragment analysis mastermix

calculations can also be performed using the autocalculate box at the

bottom of the PCR/CEQ worksheet (see appendix PNL19-2). Vortex

briefly and place on ice.

Reagents Frag. anal. mastermix

CEQ SLS buffer 20 µl x (# samples +3)=

CEQ 600 bp size standard 0.08 µl x (# samples +3)=

4.9.1.8. Place a Beckman 96-well sample plate in a cold block. Add two

drops of separation buffer to each well of the column 1 (conditioning




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lane). Next, aliquot 20 l of the prepared fragment analysis

mastermix to the required number of wells. Cover the plate loosely

with Parafilm.

4.9.1.9. Using the 10 l multichannel pipettor, add 1 l of 1:60 diluted

PCR reactions to the appropriate columns in the Beckman sample

plate. Keep sliding the Parafilm sheet from column to column to keep

track of the sample order.

4.9.1.10. Briefly (15 s) spin down the CEQ Beckman sample plate to

remove any air bubbles.

4.9.1.11. Overlay the wells containing samples with 1 drop of mineral oil.

4.9.1.12. Select “Start Sample Plate” from the “Run” pull-down menu or

click on the “Run” button in the toolbar.

4.9.1.12.1. For the CEQ 8000, a “Select the Sample Plate to Run”

window will open. Select the plate layout that was saved above.

The “Confirm Configuration-(Sample Plate Name)” window will

pop up. Confirm that the plate layout matches with the lanes

highlighted in the plate configuration window and click on “Load

Plates”. When the “Access Plates” dialog box appears, click on

“Start”. Wait for the CEQ to display the green “Go” signal box.

4.9.1.12.2. For the GeXP, a “Sample Plate Run Confirmation” window

will open. This offers the option to designate operator’s name.

At the bottom of the window, select “Sample Plate Name” and

select the plate layout that was previously saved above. Confirm

that the plate layout matches with the lanes highlighted in the

plate configuration window and that the plate location is correct.

If running two plates, designate which plate to run first by

selecting the corresponding radial button. (This window also

offers the option to replenish the gel cartridge at this time by

selecting the “Install Gel Cartridge” button at the bottom of the

window and follow instructions as previously described in

4.8.1.12). Click on “Load Plates”. When the “Access Plates”

dialog box appears, click on “Start”. Wait for the GeXP to

display the green “Go” signal box.

4.9.1.13. Lift the sample access cover. Rinse and refill wetting tray(s) with

dH2O. (If setting up on the GeXP the corresponding wetting tray for

each rail must be filled if running two plates.) Dry off outer tray,

clean off any dried deposits of gel from the lid and place the wetting

tray(s) back on machine.

4.9.1.14. Load the buffer plate(s) on the front tray and the sample plate(s)

on the back tray with notched corners top right. Close the sample

access cover. Click on “Load”. This will take you back to the

“Sample Plate Run Confirmation” window.

4.9.1.15. Once the instrument has loaded the plates, the “Start” button will

be highlighted and can be clicked to start the run.




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4.9.1.16. Running one column (8 wells) takes about 105 min. A full plate

run takes 19½ h. At end of the run, the data will be automatically

analyzed and saved.

4.10. Data export from the Beckman Coulter CEQ 8000/ 8800/GeXP

4.10.1. Day 2

4.10.1.1. To view the analyzed data, click on the “Fragments” icon in the

main CEQ window and a window labeled “Study” will open up.

4.10.1.1.1. If the “Study” window does not pop up, select “New Study”

from the “File” drop-down menu.

4.10.1.2. Select the “Analyzed Results” button and then select the desired

plate run listed in the window and click “OK”. The “Select Result

Data” window will open.

4.10.1.3. In the “Select Result Data” window, select the “Plate View” tab

and the plate format will pop up. Select the desired wells for viewing

from the plate by either clicking on them individually or select a

whole column by clicking on the column number. Selected wells will

be highlighted. Complete the sample selection by clicking on the

“Finish” button. “Fragment Analysis – New Study” window will

open with all the selected wells listed under the “Results” section.

4.10.1.4. Check the fragment result data (the fluorescent peaks) for each

well by clicking on the well IDs individually.

4.10.1.4.1. Make sure that all eight VNTRs amplified in the EDL933

control and that the fragment sizes are within the range specified

in the appendix PNL19-2 and record the fragment sizes on the

PCR/CEQ worksheet.

4.10.1.4.2. The size calling for the internal ladder should also be within

the range specified in the appendix PNL19-2 (or PNL19-5).

4.10.1.4.3. Write down any failed reactions in the PCR/CEQ worksheet.

Make a note of non-specific peaks, primer-dimers, and atypical

appearance of the DNA size standard peaks.

4.10.1.4.4. If the molecular size standard does not run all the way till the

end (640 bp) or if it skips fragments, a reaction is considered “a

fragment analysis failure” and should be re-run.

4.10.1.4.5. Low fluorescence level background peaks can sometimes

interfere with data analysis in BioNumerics. They can be filtered

out by using the following procedure:

4.10.1.4.5.1 From the “Study explorer” field on the top left corner,

click on “Fragment list”. “Exclusion filter set” is located

in the right top corner.

4.10.1.4.5.2From the “Name” drop-down menu, select the “pk height

(rfu)” option.

4.10.1.4.5.3From the “Operators” drop-down menu select the “<”

option. Type in “1000” (or the fluorescence unit value you

have determined is adequate to filter out the background




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without eliminating the true positive peaks) in the

“Value(s)” field.

4.10.1.4.5.4Click on “Apply”.

4.10.1.5. Save the study: from “File” drop-down menu, select the option

“Save Study As”. “Save Study As” dialog box will appear. Name the

study with the standardized run name (for example, “GA070426”)

and click “OK”. This ensures that you don’t have to re-analyze the

samples if you want to go to check the raw data again later.

4.10.1.6. From the “File” drop-down menu, select the option “Export

Fragments/Genotypes”. “Export Fragments/Genotypes” dialog box

will appear. Choose the location you would like the file to be saved to

(examples: E:/Compact Disk or F:/Removable Disk). Name the peak

file containing the fragment list with the run name (see section

4.8.1.7. for instructions; for example “GA070426”), make sure that

the file type is “CSV (comma limited)” and save it by clicking on

“Save” button. “Export Status” dialog box will appear. Once the

export is completed, “OK” button will be highlighted and can be

clicked.

4.10.1.7. The remaining gel can stay in the instrument if it is going to be

used within 48 hours. The shelf-life of the gel at the room

temperature is 72 hours. Using an expired gel may cause inaccurate

sizing of fragments in some loci.

5. FLOW CHART:

6. BIBLIOGRAPHY:

6.1. Hyytiä-Trees, E., Smole, S. C., Fields, P. I.., Swaminathan, B., and Ribot, E. M. (2006)

Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-

number tandem repeat analysis (MLVA) of Shiga toxin-producing Escherichia coli O157

(STEC O157). Foodborne Pathog. Dis. 3, 118-131.

6.2. Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E. (2010) Multi-laboratory

validation study of standardized multiple-locus VNTR analysis (MLVA) protocol for

Shiga toxin-producing Escherichia coli O157 (STEC O157): a novel approach to

normalize fragment size data between capillary electrophoresis platforms. Foodborne

Path. Dis. 7, 129-136.

7. CONTACTS:

7.1 Eija Trees, D.V.M., Ph.D.

PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC

(404) 639-3672

[email protected]

7.2 Patti Lafon


(404) 639-2828

mailto:[email protected]




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[email protected]

7.3 Ashley Sabol


(404) 639-2947

[email protected]

8. AMENDMENTS:

1/27/2009: Separation buffer is to be used for conditioning instead of distilled water (step 4.9.1.8)

10/6/2009: statement was added to the step 4.10.1.7. that using expired gel may cause inaccurate

fragment sizing.

6/8/2011: Instructions for operating the new GeXP version of the Beckman Coulter

Genetic Analysis system were added.

4/10/2012: “Frag-test” CEQ running conditions were replaced with “Frag-test_pause”

conditions

4/4/2013: former appendix PNL19-4 (BioNumerics specifications for the E. coli O157

VNTR loci) was moved to SOP PND14 (PulseNet standard operating procedure for

analysis of MLVA data of Shiga toxin-producing Escherichia coli in BioNumerics –

Beckman Coulter CEQ 8000/8800/GeXP data). Former appendices PNL19-5 and PNL19-

6 were renamed PNL19-4 and PNL19-5, respectively.






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Appendix PNL19-1

STEC O157 MLVA PCR Primers

Locus1 Dye2 Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’)

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

R1

VNTR-3 D2 GG CGG TAA GGA CAA CGG GGT GTT TGA ATT G GAA CAA CCT AAA ACC CGC CTC GCC ATC G

VNTR-34 Quas670 GA CAA GGT TCT GGC GTG TTA CCA ACG G GTT ACA ACT CAC CTG CGA ATT TTT TAA GTC CC

VNTR-9 Quas670 GC GCT GGT TTA GCC ATC GCC TTC TTC C GTG TCA GGT GAG CTA CAG CCC GCT TAC GCT C

VNTR-25 Quas705 GC CGG AGG AGG GTG ATG AGC GGT TAT ATT TAG TG GCG CTG AAA AGA CAT TCT CTG TTT GGT TTA CAC GAC

R2

VNTR-17 D2 GC AGT TGC TCG GTT TTA ACA TTG CAG TGA TGA GGA AAT GGT TTA CAT GAG TTT GAC GAT GGC GAT C

VNTR-19 Quas670 GC AGT GAT CAT TAT TAG CAC CGC TTT CTG GAT GTT C GGG GCA GGG AAT AAG GCC ACC TGT TAA GC

VNTR-36 Quas670 GG CGT CCT TCA TCG GCC TGT CCG TTA AAC GCC GCT GAA AGC CCA CAC CAT GC

VNTR-37 Quas705 GC CGC CCC TTA CAT TAC GCG GAC ATT C GCA GGA GAA CAA CAA AAC AGA CAG TAA TCA GAG CAG C

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

1 Primer sets are listed as multiplex PCR cocktails. 2 Wellred dye used for D2 (black); Quas705 and Quas670 used as more inexpensive replacements for Wellred D3 (green); and D4 (blue). Only the 5’ end of the forward primer is labeled.




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Note: This is posted on the WebBoard as an Excel file so users may save it locally, enter data and use the auto calculate function




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Appendix PNL19-4

Maintenance of the Beckman Coulter CEQ 8000/ 8800/GeXP

After each run

1. Clean the wetting tray thoroughly by first rinsing it with warm tap water and then with

distilled water. Remove any tried gel from the tray and the lid with a paper towel.

Refill the tray with distilled water.

2. The remaining gel can stay in the instrument if it is going to be used within 48 hours.

The shelf-life of the gel at the room temperature is 72 hours.

Instrument is not used for a week or more

1. Refill the wetting tray at least twice a week with distilled water to keep the capillaries

moist at all times.

2. It is recommended to keep a gel in the injection pump at all times to keep the pump

moist. Use leftover gels (< 3.0 ml) that cannot be used for a run anymore.

3. Make a manual purge (0.1 ml 4 times) once a week to keep the lines open and the

capillaries unblocked.

4. Remove the capillary array if the instrument is not used for more than two weeks.

Instructions to change the Beckman CEQ capillary array

1. Open the new capillary array package and carefully work loose the plastic covers from

the electrode block, but leave the covers still attached.

2. To release the old capillary array, select the “Release Capillary Array” from the “Run”

pull down menu or click on the capillary array picture in the direct control screen →

the “Remove Capillary Array” dialog box will appear

3. Open the sample access cover

4. Open the capillary access cover

5. To open the capillary temperature control cover, unlatch the two rubber latches

6. To open the manifold access cover and the plenum assembly, loosen the captive

screws

7. Lift the red “eject lever” to release the array fitting

8. Pull out the array fitting with your right hand and the electrode block with your left

hand and set aside

9. Remove the plastic covers from the electrode block and the array fitting of the new

array. Be careful not to break the capillaries!

10. Remove the yellow lens cover from the array fitting. Be careful not to touch the lens!

11. Align the array fitting and the electrode block with the guide pins.

12. Replace the manifold access cover and the plenum assembly. Carefully route the

capillaries through the whole in the plenum assembly.

13. Close the capillary temperature control cover.

14. Lower the capillary access cover and the sample access cover to their locking positions




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15. Click on “OK” in the “Remove Capillary Array” dialog box → the “Install Capillary

Array” dialog box will appear

16. Change the serial number and click first on “Set to New” and then on “Done” → the

“Confirm Capillary Array Selection” dialog box will appear → select “Yes”

17. Make sure that the instrument has a gel cartridge and a wetting tray in place. Select

“Capillary Fill” from the “Direct Control” drop-down menu. Let the instrument fill the

capillaries (take’s about 3.7 min).

18. Repeat the capillary fill step.

19. Perform three manual purge cycles with 0.1 to 0.4 ml of gel by selecting “Manifold

Purge” from the “Direct Control” drop-down menu or by clicking on the funnel on the

direct control screen.

20. To perform the optical alignment, either select “Optical Alignment” from the “Direct

Control” drop-down menu or click on the lens picture in the direct control screen

21. After the optical alignment has been completed, monitor the fluorescence baseline:

select “Monitor Baseline” from the “Run” drop-down menu → “Monitor Baseline”

dialog box will appear → check the “Enable Monitor Baseline” box and click “OK”

22. To view the baseline trace, switch to data monitor window and view the baseline for

each capillary. Baseline should be less than 5000 units. If it is higher, perform the

following procedure:

a. Release the capillary array and open the sample access cover, the capillary access

cover, the temperature control cover and the manifold access cover

b. Remove the array fitting

c. Carefully wipe clean both sides of the optical window in the array fitting by using a

cotton swab moistened with distilled water. Wipe to one direction only.

d. Dry the optical window using a second swab by wiping to the same direction as

above

e. Clean the manifold access area first by using a moist swab and then with a dry swab

f. Replace the array fitting in the manifold

g. Click on “OK” in the “Remove Capillary Array” dialog box → the “Install

Capillary Array” dialog box will appear

h. Select “Clean Capillaries” button, and click on “Done”

i. Perform two capillary fills and three manual purges

j. Repeat the optical alignment step. If the baseline is still above 5000 units after the

repeated optical alignment, the new capillary array is most likely defective and must

be replaced with another array.

23. To stop the baseline monitoring, go back to the “Monitor Baseline” dialog box and

uncheck the “Enable Monitor Baseline” box and click on “OK”

24. To check the optical scan data, go the Main menu and click on the “Sequencing” icon.

From the “File” drop-down menu, select “Open” → the “Open” dialog box will appear

→ select the “Optical scan data” tab. Find today’s optical scan file by using the filter

(type in today’s date as a start and an end date and click on “Refresh”) → highlight

today’s scan file and click on “OK”. The red and blue peaks in the optical scan data

should start from the galvanometer position 45-50 and end at about 200-210. If the

peaks start much earlier or later, the optical alignment of the instrument is off and may

contribute to increased fragment analysis failure rate. The red and blue peaks should be

superimposed at each galvanometer reading. If they are clearly separated, the optical

alignment is not optimal. In either case, a service call to the Beckman technical

support is needed.




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Instructions to make a new working database

1. Close off all open modules.

2. In the main menu of the CEQ software, click on the “Database” icon.

3. From the “File” drop-down menu, select “New Database” → The “New Database”

dialog box will appear

4. Enter a name for the new database by indicating the date on which it was created. For

example: Oct_6_2006.

5. Check the box “Set as a Working Database” and click “OK”.




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Appendix PNL19-5

Instructions to prepare the internal ladder

1. Prepare DNA templates from isolates EC04PN0139 and EC04PN0570 as described in the

protocol step 4.5. Store the templates at -20oC or -80

oC freezer.

2. Use the DNA templates to set up and run the PCR reactions R1 and R2 as described in the

protocol step 4.6.

3. After the PCR amplification, pool the R1 and R2 reactions for isolates EC04PN0139 and

EC04PN0570 into one single PCR tube to gain a total volume of 40 µl.

4. Each new lot of internal ladder must be tested against the old ladder lot by running them in the

same fragment analysis run.

5. Store the ladder in -20oC or -80

oC freezer. It should remain stable at least 5-6 freeze-thaw

cycles for a period of one month.

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)

present in the internal ladder as seen in the electropherogram from left to right:

VNTR-25 (D3): 122-124 134-136

VNTR-17 (D2): 149-152 175-178

VNTR-36 (D4): 172-175

VNTR-37 (D3): 194-197 200-203

VNTR-34 (D4): 224-227 260-262

VNTR-19 (D4): 296-299 320-322

VNTR-3 (D2): 392-395 429-431

VNTR-9 (D4): 518-521 566-570

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)

present in the internal ladder as listed in the peak file:

VNTR-17 (D2): 149-152; 175-178

VNTR-3 (D2): 392-395; 429-431

VNTR-25 (D3): 122-124; 134-136

VNTR-37 (D3): 194-197; 200-203

VNTR-36 (D4): 172-175

VNTR-34 (D4): 224-227; 260-262

VNTR-19 (D4): 296-299; 320-322

VNTR-9 (D4): 518-521; 566-570

NOTE: fragment sizes for the internal ladder based on independent runs on multiple CEQ instruments

at CDC and in PulseNet Participating Laboratories

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